The &#946;-hemoglobinopathies sickle cell disease and &#946;-thalassemia are among the most common human genetic disorders worldwide. Hemoglobin A2 (HbA2, &#945;2&#948;2) and fetal hemoglobin (HbF, &#945;2&#947;2) both inhibit the polymerization of hemoglobin S that results in erythrocyte sickling. Expression of erythroid Kruppel-like factor (EKLF) and GATA1 is critical for transitioning hemoglobin from HbF to hemoglobin A (HbA, &#945;2&#946;2) and HbA2. The lower levels of &#948;-globin expression compared with &#946;-globin expression seen in adulthood are likely due to the absence of an EKLF-binding motif in the &#948;-globin proximal promoter. In an effort to upregulate &#948;-globin to increase HbA2 expression, we created a series of EKLF-GATAl fusion constructs composed of the transactivation domain of EKLF and the DNA-binding domain of GATAl and then tested their effects on hemoglobin expression. EKLF-GATAl fusion proteins activated &#948;-, &#947;-, and &#946;-globin promoters in K562 cells, and significantly upregulated &#948;- and &#947;-globin RNA transcripts and proteins expression in K562. We found that the expression of long-form EKLF-GATA1 increased &#948;-, &#947;-, and &#946;-globin promoter activity 1.7-, 2.2-, and 6.8-fold, respectively, at 24 hours after transfection, and 5.4-, 2.9-, and 9.4-fold, respectively, at 48 hours after transfection (when compared with mock transfection). The effect of medium-form EKLF-GATA1 expression on globin promoter activity was less profound than that of long-form EKLF-GATA1, with a 1.9-fold increase for &#947;-globin promoter activity at 24 hours and a 2.5- and 3.2-fold increase for &#948;- and &#947;-globin promoter activity, respectively, at 48 hours. Both the short-form EKLF-GATA1 and vector only had no appreciable effect on globin promoter activity after 24 or 48 hours. GATA1 expression increased &#948;-globin promoter activity approximately 2-fold at 24 hours and 4.3-fold at 48 hours; EKLF induced &#946;-globin promoter activity approximately 2-fold at both 24 and 48 hours. These results indicate the long- and medium-form of EKLF-GATA1 fusion proteins, which contain the N-finger and C-finger of the GATA1-binding domain, may well bind to and activate the &#948;-globin promoter. In contrast, the short-form of EKLF-GATA1 fusion protein, which lacked the intact C-finger, was not able to bind to the &#948;-globin promoter and thus had no impact on globin expression. In CD34+ cells, tThe long-form EKLF-GATA1 upregulated &#946;-globin expression 1.7-fold, &#948;-globin gene expression 2.7-fold, and &#947;-globin gene expression 1.9-fold. The medium-form EKLF-GATA1 upregulated &#948;-globin gene expression 2.2-fold and &#947;-globin 1.3-fold, but had no effect on &#946;-globin gene expression. We also observed that EKLF only-transduced CD34+ cells expressed 1.4-fold higher levels of &#946;-globin expression, and GATA1 only-transduced cells expressed 1.5-fold higher levels of &#948;-globin and 1.3-fold higher levels of &#946;-globin. In contrast, the short-form of EKLF-GATA1 had no significant effect on globin expression. The results of gene expression were confirmed in both K562 and CD34+ cells, in Western Blot analysis. The binding of EKLF-GATA1 fusion proteins at the GATA1 consensus site in the &#948;-globin promoter was confirmed by chromatin immunoprecipitation assay. In summary, we present two functional EKLF-GATA1 fusion proteins containing the GATA1 primary binding domain that could bind to and activate &#948;-globin promoter and significantly increase &#948;-globin expression in K562 cells and CD34+ bone marrow cells. Although the long-form EKLF-GATA1 fusion protein also increased &#946;-globin expression in CD34+ cells, its major effects were on &#948;- and &#947;-globin induction; the medium-form EKLF-GATA1 elevated &#948;- and &#947;-globin expression without an effect on &#946;-globin expression. Induction of both &#948;- and &#947;-globin expression may be beneficial for an antisickling effect and compensating for impaired &#946;-globin production. These EKLF-GATA1 fusion proteins could prove useful as a genetic therapeutic tool for SCD and &#946;-thalassemia, and warrant further preclinical evaluation in vivo.